Current Gene Therapy - Volume 20, Issue 5, 2020

Adeno-associated virus (AAV) is a promising vector for in vivo gene therapy because of its excellent safety profile and ability to mediate stable gene expression in human subjects. However, there are still numerous challenges that need to be resolved before this gene delivery vehicle is used in clinical applications, such as the inability of AAV to effectively target specific tissues, preexisting neutralizing antibodies in human populations, and a limited AAV packaging capacity. Over the past two decades, much genetic modification work has been performed with the AAV capsid gene, resulting in a large number of variants with modified characteristics, rendering AAV a versatile vector for more efficient gene therapy applications for different genetic diseases.

Respiratory diseases are one of the prime topics of concern in the current era due to improper diagnostics tools. Gene-editing therapy, like Clustered regularly interspaced palindromic repeats- associated nuclease 9 (CRISPR/Cas9), is gaining popularity in pulmonary research, opening up doors to invaluable insights on underlying mechanisms. CRISPR/Cas9 can be considered as a potential gene-editing tool with a scientific community that is helping in the advancement of knowledge in respiratory health and therapy. As an appealing therapeutic tool, we hereby explore the advanced research on the application of CRISPR/Cas9 tools in chronic respiratory diseases such as lung cancer, Acute respiratory distress syndrome (ARDS) and cystic fibrosis (CF). We also address the urgent need to establish this gene-editing tool in various other lung diseases such as asthma, Chronic obstructive pulmonary disease (COPD) and Idiopathic pulmonary fibrosis (IPF). The present review introduces CRISPR/Cas9 as a worthy application in targeting epithelial-mesenchymal transition and fibrinolytic system via editing specific genes. Thereby, based on the efficiency of CRISPR/Cas9, it can be considered as a promising therapeutic tool in respiratory health research.

The ovary serves as the source of oocytes for the maintenance of female fertility and is a major supplier of sex hormones for endocrine homeostasis. Various circumstances such as genetic defects, autoimmune disorders, natural aging and environmental toxins can damage the ovaries leading to diminished ovarian function, and there are currently no effective treatment regimens for such loss of function. Stem cells show promise for treating many refractory diseases, and stem cell transplantation has been shown to be effective and safe as a new therapeutic method for ovarian injuries and ovarian aging in both animal models and women with premature ovarian insufficiency. However, the specific mechanisms that underlie the observed positive outcomes of improving ovarian function are not well understood. Evidence is mounting that stem cell-derived conditioned medium, exosomes, and trophic growth factors can also inhibit ovarian damage and alleviate the age-related fertility decline in female mice, indicating that stem cells exert the paracrine effects. Further studies to elucidate the cellular and molecular mechanisms, including signaling pathways, for improving ovarian function and promoting the secretory capacity of stem cells will fill the bench-to-bedside gap of stem cell therapy in the clinic. Furthermore, in-depth analyses of the stem cell secretome and identification of the key effective components will underlie a new paradigm in cell-free therapeutic strategies for ovarian insufficiency and ovarian aging.

In recent years, RNA interference technology has been extensively studied for its therapeutic potential against a wide variety of diseases. It aims to silence the expression of undesired genes associated with the target disease by the administration of RNA interference agents. However, these agents (nucleic acids) are unstable in the circulatory system and lack target specificity. Drug delivery systems are, therefore, crucial for the successful practice of the technique. A wide array of delivery systems has been developed to conquer these challenges, such as viral vectors, inorganic drug carriers, polymeric carriers and lipid-based carriers, with, however, significant limitations. In addition to the existing technologies, novel, innovative drug delivery systems, such as the configurable xenobot, are emerging at a rapid pace and have the potential to take the realm of biomedicine to the next level. This review summarizes technical difficulties in the development of drug delivery systems and current technologies developed for delivering RNAi agents with a discussion on their limitations.

Ischemic stroke is one of the main causes of mortality in advanced societies. Although gene therapy can be helpful, delivering gene therapy agents is challenging. Nanotechnology can enhance the potential therapeutic effects and the efficiency of gene therapy for some brain disorders. The present systematic review was conducted based on the PRISMA protocol to investigate the possible therapeutic effects of nanoparticles as the carriers of gene therapy agents in stroke therapy. Relevant keywords were used to search from ISI Web of Science, PubMed, and Scopus for relevant publications up to April 24, 2020. The selected articles were assessed using certain scores on the quality of the articles. Data extraction and quality judgment were carried out by the present reviewers. Of 130 articles retrieved, seven met the inclusion criteria and were, therefore, included in the final analysis. The outcome of the reviewing process revealed that depending on the selection of the target genes, stroke gene therapies have acceptable therapeutic consequences. The nanoparticles could be used to carry the gene therapy agents that are efficient targeting in stroke treatment. Also, it appears that the use of nanoparticles such as PEGylation and PAMAM, can be a valuable option to intensify the efficiency and specific targeting of stroke location. In conclusion, due to the inability of brain regeneration and the importance of genes in stroke-related complications, gene therapy seems to be a suitable treatment strategy. The use of suitable nanoparticles for transportation ensures the efficiency and usefulness of this method.

Background: Lipopeptide-based gene carriers have shown low cytotoxicity, are capable of cell membrane penetration, are easy to manufacture and therefore are great potential candidates for gene delivery applications. Objectives: This study aims to explore a range of short synthetic lipopeptides, (Lau: Lauryl; Pal: Palmitoyl) consisting of an alkyl chain, one cysteine (C), 1 to 2 histidine (H), and lysine (K) residues by performing in-silico molecular interaction and in-vitro evaluation. Methods: The molecular interactions between the lipopeptides and Importin-α receptor were performed using AutoDock Vina and Amber14. The lipopeptide/DNA complexes were evaluated in- -vitro for their interactions, particle size, zeta potential and transgene expression. Transfection efficiency of the lipopeptides and Pal-CKKHH-derived liposome was carried out based on luciferase transgene expression. Results: The in-silico interaction showed that Lau-CKKH and Pal-CKKHH hypothetically expedited nuclear uptake. Both lipopeptides had lower binding energy (-6.3 kcal/mol and -6.2 kcal/mol, respectively), compared to the native ligand, viz, nuclear localization sequence (-5.4 kcal/mol). The short lipopeptides were able to condense DNA molecules and efficiently form compacted nanoparticles. Based on the in-vitro evaluation on COS-7, Pal-CKKHH was found to be the best transfection agent amongst the lipopeptides. Its transfection efficiency (ng Luc/mg total protein) increased up to ~3-fold higher (1163 + 55) as it was formulated with helper lipid DOPE (1:2). The lipopeptide- based liposome (Pal-CKKHH: DOPE=1:2) also facilitated luciferase transgene expression on human embryonic kidney cells (293T) and human cervical adenocarcinoma cells (HeLa) with transfection efficiency 1779 +52 and 260 + 22, respectively. Conclusion: Our study for the first time has shown that the fully synthesized short lipopeptide Pal- CKKHH is able to interact firmly with the Importin-α. The lipopeptide is able to condense DNA molecules efficiently, facilitate transgene expression, expedite the nuclear uptake process, and hence has the characteristics of a potential transfection agent.

Background: Recruitment of gene modifying bone marrow mesenchymal stem cells (BMSCs) has been considered an alternative to single-cell injection in articular cartilage repair. Purpose: This study aimed to investigate whether the effect of runt-related transcription factor 2(Runx2) overexpression bone marrow mesenchymal stem cells in vivo could improve the quality of repaired tissue of a knee cartilage defect in a rabbit model. Methods: Thirty-two New Zealand rabbits were randomly divided into four groups. The blank group (Con) did not receive anything, the model group (Mo) was administered saline, the simple stem cell group (MSCs) received MSCs injection, and the Runx2 transfection group (R-MSCs) received Runx2 overexpression MSCs injection. After adapting to the environment for a week, a 5 mm diameter cylindrical osteochondral defect was created in the center of the medial femoral condyle. Cell and saline injections were performed in the first and third weeks after surgery. The cartilage repair was evaluated by macroscopically and microscopically at 4 and 8 weeks. Results: Macroscopically, defects were filled and surfaces were smoother in the MSCs groups than in the Mo group at 4th week. Microscopically, the R-MSCs group showed coloration similar to surrounding normal articular cartilage tissue at 8 weeks in masson trichrome staining. The COL-II, SOX9, and Aggrecan mRNA expressions of MSCs were enhanced at 4 weeks compared with R-MSCs, then the expression reduced at 8 weeks, but was still higher than Mo group level (P<0.05). The western blot examination revealed that the COL-IIand SOX9 expression of MSCs was higher than R-MSCs at 4 weeks, then the expression reduced at 8 weeks, but was still higher than the Mo level (P<0.05). The IL-1β content in the joint fluid also revealed that cartilage repair with R-MSCs was better than that with MSCs at 8 weeks (P<0.05). Conclusion: The R-MSCs group showed cellular morphology and arrangement similar to surrounding normal articular cartilage tissue, and Runx2 overexpression of MSCs resulted in overall superior cartilage repair as compared with MSCs at 8 weeks.